In our investigation of the factors that control the assembly of tubulin into microtubules and the distribution of microtubules in the cell, we have focused this year on the electrostatics of vinblastine interactions with tubulin and the formation of abnormal spiral polymers formed by the drug. Unlike microtubule assembly, vinblastine-induced polymerization does not require GTP but is inhibited by this and other nucleoside triphosphates and by inorganic oligophosphates.(P3<P2<P). This appears to be, in part an effect on nucleation since addition of microtubule seeds overcomes the inhibition by anions and reduces the latent period for the onset of polymerization (Eur. J. Biochemistry 250: 425-431, 1997). We conclude that charge-charge interactions play a significant role in vinblastine-induced polymer formation. Removal of the highly anionic carboxyl termini (see Biochemistry 37: 10722-10729, 1998) for the cooperative charge interactions of these C termini) of beta but not alpha tubulin by limited proteolysis with subtilisin attenuates the GTP effect and enhances vinblastine-induced polymerization. The vinblastine effect titrates over a narrow pH range (6.5-7.3) suggesting the involvement of one or more histidine residues (Proc. Natl. Acad. Sci. USA 95; 4253-4257). Modification of the most reactive His residue of tubulin with diethylpyrocarbonate at a mole ratio of 0.74 to total His residues completely inhibits vinblastine-induced polymerization, with little effect on microtubule assembly. Only two His residues are modified, and these are located on beta tubulin at His 227 and at His 264. These modifications are reversible with recovery of polymerization competence by use of hydroxylamine. By contrast, inhibition of microtubule assembly requires modification of two additional His residues; only one of which could be located, at His 88 of alpha tubulin. Thus while histidine residues are involved in both microtubule assembly and vinblastine-induced polymerization, these occur at different sites and on different monomers (J. Biol. Chem. 273:33131-33137, 1998). We are continuing in the idenfication of the palmytoylations sites in both alpha and beta tubulin and the factors regulating palmitoylation (Biochem. Biophys. Res. Commun. 239:650-654). Alpha and beta tubulin are differentially regulated and it appears that there are substantial differences in the lability or reversibility of the palmitc acid bonds to tubulin. The labile ones are consistent with thioester bonds but the more hydroxylamine-resistant bonds may be either buried thioesters, or oxyesters. Detergent distribution studies in two-phase systems suggest substantial hydrophobicity despite the high charge density of tubulin.